Reactivation of HSV-1 from latency is associated with many disease states ranging from benign cold sores to life-threatening encephalitis. Therefore, understanding the mechanism that controls the switch from the latent to the lytic phase of HSV-1 infection is an important public health priority. This proposal addresses the role of oniL, a unique origin of replication of HSV-1, in the biology of the virus during productive infection and reactivation from latency. Technology will be developed to introduce point mutations into selected regions of oriL thought to be important for the initiation of origin-dependent DNA replication and wild-type and mutated origins will be compared for replication efficiency in transient DNA replication assays in cells of neural and non-neural lineage. Mutations that affect oriL-dependent DNA Replication in vitro will be introduced into the viral genome and the resulting mutant viruses will be studied in cells of neural and non- neural lineage with respect to viral growth, viral DNA replication, and transcription of several viral genes including those that flank oriL and oriS. Wild-type and oriL mutant viruses will be further characterized in vivo in a mouse ocular model during acute infection and reactivation from latency to determine the role of oriL in the pathogenesis of HSV-1.